Seroprevalence of adeno-associated virus types 1, 2, 3, 4, 5, 6, 8, and 9 in a Basque cohort of healthy donors

Adeno-associated viruses (AAVs) are promising gene therapy vectors, but challenges arise when treating patients with preexisting neutralizing antibodies. Worldwide seroprevalence studies provide snapshots of existing immunity in diverse populations. Owing to the uniqueness of the Basque socio-geographical landscape, we investigated the seroprevalence of eight AAV serotypes in residents of the Basque Country. We found the highest seroprevalence of AAV3, and the lowest seroprevalence of AAV9. Additionally, less than 50% of the Basque population has neutralizing antibodies against AAV4, AAV6, and AAV9. Our findings provide insight into AAV infections in the Basque region, public health, and the development of AAV-based therapeutics.

www.nature.com/scientificreports/gathered insights provided relevant information on which serotypes are more commonly circulating across different locations worldwide.
The Basque Country is a region that spans parts of northern Spain and southwestern France.In recent years, autochthonous Basque inhabitants have garnered significant research attention due to their distinctive cultural, biological and genetic background from other European populations 16,17 .However, according to the Basque Institute of Statistics (EUSTAT), as of January 2022, nearly 30% of the current residents of the Basque Country were born in the remaining Spanish provinces and to foreigners (https:// en.eustat.eus/ indice.html).
We sought to target a healthy cohort of Basque country residents for the presence of NAbs against AAVs to assess their frequency and cross-reactivity.We showed that neutralizing antibodies against AAV3 were the most prevalent antibodies in our cohort.In addition, using a 95% confidence interval, we extrapolated that less than 50% of Basque residents possess NAbs against the AAV4, AAV6 and AAV9 serotypes.Finally, it is possible to statistically detect differences in the NAb seroprevalence for the AAV5, AAV8 and AAV9 serotypes in the European/Mediterranean landscape, even though the prevalence of these serotypes was opposite to that in countries geographically close such as France or Italy.

Co-seroprevalence of neutralizing antibodies
Seroreactivity between AAV serotypes provides valuable information for inferring which serotype would be more advantageous for development or administration than other serotypes.All sera positive for NAbs against AAV1 showed correlation with almost all other serotypes.All samples that were NAbs-positive against AAV1 were also NAbs-positive against AAV2.Furthermore, samples NAbs-positive against AAV1 had a 97.6% chance of having NAbs against AAV3, 83.3% against AAV6, 76.2% against AAV5 and 71.4% against AAV8.A correlation of 31% in NAbs presence was found between AAV1 and AAV9.
Finally, only two serotypes, AAV2 and AAV3, were neutralised with fourteen serum samples (82.4%) that were NAb-positive against AAV9.The above results are summarised in Fig. 2.

Comparison of seroprevalence between the Basque cohort and cohorts from European countries
Previous studies have provided an epidemiological map of NAbs against AAVs in ten different countries across four continents, although not all using the eight serotypes as in our study 10,13 .We compared the seroprevalence observed in the Basque cohort (n = 100) with that reported in France (n = 152), Belgium (n = 100), Italy (n = 100) and Greece (n = 81).These studies, as well as those worldwide were performed with equivalent serum dilution (1:20) 10,13 .To the best of our knowledge, no seroprevalence study of AAV3 under similar methodological conditions is available for any of the other European cohorts.AAV8 was the only serotype showing a significant difference in seroprevalence when compared to all European cohorts; the Basque cohort showed a 52% NAb seroprevalence, whereas Belgium had 32%, Greece 21%, France 18% and Italy 13%.As previously shown, AAV2 is the serotype that has the highest seroprevalence in the EU and worldwide, whereas in our cohort of Basque residents, AAV3 exhibits the highest seroprevalence.Additionally, our data underline a significant discrepancy in seroprevalence versus that found in Greece (37%) and in Italy (23%).Finally, AAV1, AAV5 and AAV9 showed only one country that differed significantly: Italy for AAV1 (13%) and France for AAV5 and AAV9 (4% and 34%, respectively) (Fig. 3A).

Basque cohort seroprevalence in the context of the worldwide epidemiology of NAbs to AAVs
We updated the European frequencies of AAV seroprevalence with our hundred samples from the Basque cohort and compared them with the available data across countries and continents (n = 100 samples for the U.S., China and Australia cohorts; n = 407 for the African cohort) 10,14 .The European seroprevalence of anti-AAV1 NAbs (37%) significantly differed from that in the U.S. (20%).Except for Australia, the seroprevalence of anti-AAV2 NAbs (44%) exhibited significant differences from the seroprevalence found in China (92%), Africa (56%) and the U.S. (28%).Moreover, the Chinese cohort showed a large increase in the seropositive profile against AAV3 (52% vs. 89%).Finally, when comparing the AAV8 seroprevalence in the EU (29%), statistically significant differences were found with those in China (69%) and the U.S. (14%) (Fig. 3B).

Discussion
Adeno-associated viruses have emerged as one of the most promising viral vectors for gene therapy.In the last six years, the U.S. FDA has approved drugs using different serotypes of recombinant AAV vectors, including AAV2, AAV5 and AAV9 (FDA submission tracking numbers 125610, 125694, 125772, 125720, 125781 and 125786, respectively, in chronological order).However, as preexisting NAbs jeopardize effective treatment 18,19 , knowing the population seroprevalence profile has practical value in a clinical setting and for AAV drug development.This study was designed to stratify the seroprevalence of the Basque population.To the best of our knowledge, no prior study of this nature has been conducted in the Basque region.We showed that the population of the Basque Autonomous Region possesses the highest seroprevalence of neutralizing antibodies against AAV3, compared to the AAV2 seroprevalence found in previous reports for cohorts in the U.S., Belgium, Greece, Italy, Kenya, Rwanda, South Africa, Uganda, Zambia, Australia, France, China and Japan 10,[12][13][14][20][21][22] . In addtion, our study revealed that, in the Basque cohort, there was a statistically significant decrease in the AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 seroprevalence versus the AAV3 seroprevalence.Moreover, less than 50% of the residents in the Basque Country possess neutralizing antibodies against AAV4, AAV6 and AAV9.In this context, these serotypes might be more suitable targets for gene therapy development.
We also observed co-seroreactivity across different AAV serotypes within the Basque cohort.Co-seroreactivity occurs when (i) an individual has been infected with different AAV serotypes throughout her/his life, thus possessing both specific NAbs, and/or (ii) a NAb raised against a specific serotype recognizes multiple serotypes.The latter difference may be explained by the structural and/or primary sequence homology of the VP1-VP2-VP3 capsid proteins across the different serotypes.An example of this could be those sera positive for neutralizing anti-AAV6 antibodies that are also positive for neutralizing antibodies against AAV1, AAV2 and AAV3 [largest structural pairwise root-mean-square-deviation (rmsd) = 0.8 Å between 518 equivalent Cα in AAV6 versus AAV3 and a minimal sequence identity > 83% between AAV6 versus AAV2; PDB ids for AAV6, AAV1, AAV2 and AAV3: 3OAH, 6JCR, 6IH9 and 3KIE, respectively].
However, this rationale has its limitations as it does not explain the significant differences, such as the low prevalence percentage observed for AAV9 among the AAV8-positive serum samples, despite displaying a rmsd of 1.1 Å between 518 equivalent Cα and a sequence identity > 85% (PDB ids for AAV9 and AAV8: 7MT0 and 6V12, respectively) 23 .It is important to consider that most NAbs interact with the receptor binding domains located at the threefold axis of symmetry on the capsid, and these interactions typically involve a few key residues conformationally competent.
Posttranslational modifications, such as glycosylation, though less common in nonenveloped viruses, remain largely unexplored in AAV research and might modulate NAb recognition 24 .
When analysing our results derived from the Basque cohort against those available from other EU cohorts (France, Belgium, Italy and Greece), we noticed that only seroprevalence data for AAV1, AAV2 and AAV8 were common.A comparison of the percentages of seropositivity against AAV1 showed that in the Basque cohort, there was a statistically significant increase (42%) relative to that in the Italian cohort (13%), which was more in line with the seropositivity values of other EU countries.
In the case of NAbs against AAV2, the Basque cohort (54%) had seroprevalences similar to those of the French and Belgian cohorts but significantly different from the lower percentages of the Italian (23%) and Greek (37%) cohorts.Notably, for the remaining serotypes, when we compared the Basque cohort against the French cohort (which is the closest country geographically), there were large statistically significant differences in AAV5 (42% vs. 4%) and AAV9 (17% vs. 34%) 10,13 .
When all European data were combined, AAV2 (44%) and AAV8 (29%) were the serotypes that exhibited the most significant differences, with a decrease in prevalence for both serotypes in the U.S. (28% and 14%), an increase in both serotypes in China (92% and 69%, respectively) and an increase in AAV2 in Africa (56%).Regarding the prevalence of AAV1, only the U.S. (20%) showed a decrease when compared to Europe (37%).Finally, only one previous study analysed NAbs against the AAV3 serotype, revealing that there is a large statistical increase in NAbs in China (89%) compared to Europe (52%).).The absence of panels for serotypes AAV4, AAV5, AAV6 and AAV9 is due to the unavailability of seroprevalence data.The asterisk indicates statistical significance compared to the corresponding NAb incidence serotype from the European cohort (p value ≤ 0.05).

Figure 1 .
Figure 1.A Seroprevalence of neutralizing antibodies (NAbs) against different adeno-associated virus (AAV) serotypes in 100 healthy human serum samples from residents of the Basque Country.Samples were considered positive if the 1:20 serum dilution reduced the vector transduction ≥ 50%.The asterisk indicates statistical significance (p value ≤ 0.05).B 95% confidence intervals (CIs) for neutralizing antibodies against AAVs within the Basque population.

Figure 2 .
Figure 2. Co-seroprevalence distribution of NAbs against AAV serotypes.The number in bold at the top of each cell indicates the number of sera positive for the serotype in the row among the resulting NAb-positive samples against the serotype in the column.The number in parenthesis at the bottom of each cell indicates the percentage of sera positive against the serotype in the row relative to the total number of NAb-positive sera against the serotype in the column.False discovery rate (FDR) corrected by the Benjamini-Hochberg method (cells in yellow: p value ≤ 0.05). https://doi.org/10.1038/s41598-024-66546-4

Figure 3 .
Figure 3.A Comparative seroprevalence of NAbs against AAV serotypes across European countries (up to 152 serum samples from France, 100 serum samples from Belgium, 100 serum samples from Italy and 81 serum samples from Greece; data extracted from Refs.10,13 ).The absence of panels for serotypes AAV3 and AAV4 is due to the unavailability of seroprevalence data.The asterisk marks the statistical significance when compared with the same NAb prevalence serotype from the Basque cohort (p value ≤ 0.05).B Seroprevalence profile of NAbs against different adeno-associated virus (AAV) serotypes in a worldwide comparison to date.The European cohort included 533 serum samples, the African cohort 407, and the U.S., Chinese and Australian cohorts each included 100 serum samples (data extracted from Refs.10,13,14 ).The absence of panels for serotypes AAV4, AAV5, AAV6 and AAV9 is due to the unavailability of seroprevalence data.The asterisk indicates statistical significance compared to the corresponding NAb incidence serotype from the European cohort (p value ≤ 0.05).